Glutarate Compounds for Treating Ischemia-Reperfusion Injuries

ABSTRACT

Disclosed herein are methods of preventing, inhibiting, reducing, or treating ischemia and reperfusion injury to tissues with glutarate compounds such as α-ketoglutarate.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under AT006889 andHL119886, awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to treating ischemia-reperfusion injuries.

2. Description of the Related Art

Ischemia-reperfusion injury is a common clinical problem, whetherresulting from spontaneous events (such as heart attack and stroke) orinduced artificially during surgery and organ transplantation. Standarddeclotting and antiplatelet treatments as part of the reperfusionprocedure aim to save the life of the patient, but do not address thedebilitating tissue/organ damage caused by ischemia and/or reperfusion.Ischemia-reperfusion injuries are the leading cause of morbidity andmortality after such events.

Therefore, a need exists for treatments for ischemia-reperfusioninjuries.

SUMMARY OF THE INVENTION

In some embodiments, the present invention is a method of preventing,inhibiting, reducing, or treating an ischemia-reperfusion injury (IRinjury) to a tissue in or of a subject, which comprises administering tothe tissue or the subject a therapeutically effective amount of at leastone glutarate compound before, during, and/or after an event that causesan IR injury. In some embodiments, at least one glutarate compound is acompound of Formula I or Formula II as disclosed herein. In someembodiments, the at least one glutarate compound is an α-KG compound ora 2-HG compound as disclosed herein. In some embodiments, the at leastone glutarate compound is α-ketoglutarate (α-KG), 2-hydroxyglutaricacid, and/or (S)-2-hydroxyglutaric acid (S-2HG). In some embodiments,the at least one glutarate compound is administered in the form of apharmaceutical composition. In some embodiments, the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier. In someembodiments, the at least one glutarate compound is administered within1 to 2 hours of the event that causes an IR injury. In some embodiments,the at least one glutarate compound is administered within 1 hour of theevent that causes an IR injury. In some embodiments, the at least oneglutarate compound is administered within 30 minutes of the event thatcauses an IR injury. In some embodiments, the at least one glutaratecompound is administered during the event that causes an IR injury. Insome embodiments, the at least one glutarate compound is administeredimmediately prior to the event that causes an IR injury. In someembodiments, the glutarate compound is administered after the event thatcauses an IR injury. In some embodiments, the therapeutically effectiveamount is administered as a single dose. In some embodiments, thetherapeutically effective amount is about 5-20 mg/kg or about 10-15mg/kg weight of the subject. In some embodiments, the at least oneglutarate compound is an α-KG compound, and the therapeuticallyeffective amount is about 5-15 mg/kg, about 10-15 mg/kg, or about 11-12mg/kg weight of the subject. In some embodiments, the at least oneglutarate compound is a 2-HG compound, and the therapeutically effectiveamount is about 10-20 mg/kg, about 15-20 mg/kg, or about 15-16 mg/kgweight of the subject. In some embodiments, the at least one glutaratecompound is administered directly to the tissue in vivo, for example, tocardiac tissue during surgery on the heart. In some embodiments, the atleast one glutarate compound is administered directly to the tissue exvivo, for example, by preserving, flushing, or reperfusing the tissuewith a fluid, such as preservation fluid, flush fluid, or reperfusionfluid used in organ transplantations, that comprises the at least oneglutarate compound. In some embodiments, the tissue is cardiac tissue.In some embodiments, the tissue is an organ. In some embodiments, theorgan is a heart, liver, lung, kidney, or the like. In some embodiments,the event that causes an IR injury is ischemia, reperfusion,thromboembolism, vasoconstriction, cardiac standstill, myocardialinfarction, stroke, or surgery. In some embodiments, the event is amyocardial infarction.

In some embodiments, the present invention is a method of treating orreducing an ischemia-reperfusion injury (IR injury) to a tissue in or ofa subject, which comprises administering to the tissue or the subject apharmaceutical composition comprising a therapeutically effective amountof a compound of Formula I:

wherein R¹ is hydrogen, halogen, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹,—SR¹⁰, substituted or unsubstituted alkyl, or substituted orunsubstituted heteroalkyl; R² and R³ are each independently hydrogen,halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl; or R² and R³, together with the atom to which they arebound, form an oxo; R⁴ and R⁵ are each independently hydrogen, halogen,—CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R⁶ ishalogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl; and R⁷, R⁸, R⁹, and R¹⁰ are each independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; or a salt thereof. In someembodiments, R¹ is hydrogen, —CHO, or —OR⁷. In some embodiments, R¹ is—OR⁷, wherein R⁷ is hydrogen, substituted or unsubstituted alkyl, orsubstituted or unsubstituted aryl. In some embodiments, R¹ is —OR⁷,wherein R⁷ is C₁₋₂₀ substituted or unsubstituted alkyl. In someembodiments, R² is hydrogen, halogen, —CN, —CHO, or —NR⁸R⁹, wherein R⁸and R⁹ are each independently hydrogen or substituted or unsubstitutedalkyl. In some embodiments, R² and R³, together with the atom to whichthey are bound, form an oxo. In some embodiments, R⁴ and R⁵ are eachindependently hydrogen, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹, whereinR⁷, R⁸, and R⁹ are each independently hydrogen or C₁₋₂₀ substituted orunsubstituted alkyl. In some embodiments, R⁶ is —CHO, —OR⁷, —NR⁸R⁹,—COOR⁷, or —CONR⁸R⁹, wherein R⁷, R⁸, and R⁹ are each independentlyhydrogen or C₁₋₂₀ substituted or unsubstituted alkyl. In someembodiments, R⁶ is —COOR⁷ or —CONR⁸R⁹, wherein R⁷, R⁸, and R⁹ are eachindependently hydrogen or C₁₋₂₀ substituted or unsubstituted alkyl. Insome embodiments, the compound is alpha-ketoglutaric acid (α-KG). Insome embodiments, the compound is dimethyl 2-oxoglutarate. In someembodiments, the compound is a 2-HG compound. In some embodiments, thecompound is 2-hydroxyglutaric acid. In some embodiments, the compound is(S)-2-hydroxyglutaric acid (S-2HG). In some embodiments, the compound isadministered during an event that causes an IR injury. In someembodiments, the compound is administered after the event that causes anIR injury. In some embodiments, the compound is administered within 1 to2 hours of the event. In some embodiments, the compound is administeredwithin 1 hour of the event. In some embodiments, the compound isadministered within 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10minutes, or 5 minutes of the event. In some embodiments, the compound isadministered as a single dose. In some embodiments, the therapeuticallyeffective amount is about 5-20 mg/kg, or about 10-15 mg/kg weight of thesubject. In some embodiments, the compound is administered to thesubject in combination with an additional therapeutic agent. In someembodiments, the additional therapeutic agent is oligomycin. In someembodiments, the additional therapeutic agent is aspirin. In someembodiments, the event is a myocardial infarction. In some embodiments,the tissue is cardiac tissue.

In some embodiments, the present invention is a method of preventing orinhibiting an ischemia-reperfusion injury (IR injury) to a tissue in orof a subject, which comprises administering to the tissue or the subjectpharmaceutical composition comprising a therapeutically effective amountof a compound of Formula I:

wherein R¹ is hydrogen, halogen, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹,—SR¹⁰, substituted or unsubstituted alkyl, or substituted orunsubstituted heteroalkyl; R² and R³ are each independently hydrogen,halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl; or R² and R³, together with the atom to which they arebound, form an oxo; R⁴ and R⁵ are each independently hydrogen, halogen,—CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R⁶ ishalogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl; and R⁷, R⁸, R⁹, and R¹⁰ are each independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; or a salt thereof; and anexcipient. In some embodiments, R¹ is hydrogen, —CHO, or —OR⁷. In someembodiments, R¹ is —OR⁷, wherein R⁷ is hydrogen, substituted orunsubstituted alkyl, or substituted or unsubstituted aryl. In someembodiments, R¹ is —OR⁷, wherein R⁷ is C₁₋₂₀ substituted orunsubstituted alkyl. In some embodiments, R² is hydrogen, halogen, —CN,—CHO, or —NR⁸R⁹, wherein R⁸ and R⁹ are each independently hydrogen orsubstituted or unsubstituted alkyl. In some embodiments, R² and R³,together with the atom to which they are bound, form an oxo. In someembodiments, R⁴ and R⁵ are each independently hydrogen, —CHO, —OR⁷,—NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹, wherein R⁷, R⁸, and R⁹ are eachindependently hydrogen or C₁₋₂₀ substituted or unsubstituted alkyl. Insome embodiments, R⁶ is —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, wherein R⁷, R⁸, and R⁹are each independently hydrogen or C₁₋₂₀ substituted or unsubstitutedalkyl. In some embodiments, R⁴ is —COOR⁷ or —CONR⁸R⁹, wherein R⁷, R⁸,and R⁹ are each independently hydrogen or C₁₋₂₀ substituted orunsubstituted alkyl. In some embodiments, R⁶ is —COOR⁷, wherein R⁷ ishydrogen. In some embodiments, the compound is alpha-ketoglutarate(α-KG). In some embodiments, the compound is a 2-HG compound. In someembodiments, the compound is disodium (S)-2-hydroxyglutarate (S-2HG). Insome embodiments, the compound is administered prior to an event thatcauses an IR injury. In some embodiments, the compound is administeredas a single dose. In some embodiments, the therapeutically effectiveamount is about 5-20 mg/kg, or about 10-15 mg/kg weight of the subject.In some embodiments, the tissue is cardiac tissue.

In some embodiments, the present invention is a method of increasingcyclophilin D (CypD)-Complex V formation in a cell, which comprisescontacting a cell with at least one glutarate compound. In someembodiments, at least one glutarate compound is a compound of Formula Ior Formula II as disclosed herein. In some embodiments, the at least oneglutarate compound is an α-KG compound or a 2-HG compound as disclosedherein. In some embodiments, the at least one glutarate compound isα-ketoglutarate (α-KG), 2-hydroxyglutaric acid, dimethyl 2-oxoglutarate,and/or (S)-2-hydroxyglutaric acid (S-2HG). In some embodiments, theincrease in cyclophilin D-Complex V formation is relative to a control,e.g., the formation of CypD and Complex V of an equivalent cell in theabsence of the at least one glutarate compound. In some embodiments, thecell is from a cardiac tissue. In some embodiments, the cell is from amammalian cardiac tissue. In some embodiments, the cell is from a humancardiac tissue.

In some embodiments, the present invention is directed to the use of atleast one glutarate compound in the manufacture of a medicament forpreventing, inhibiting, reducing, or treating an ischemia-reperfusioninjury (IR injury) to a tissue in or of a subject. In some embodiments,the present invention is directed to the use of at least one glutaratecompound for the manufacture of a medicament for preventing, inhibiting,reducing, or treating an ischemia-reperfusion injury (IR injury),wherein the medicament is prepared to be administered in accordance withone or more of the dosage regimens as described herein, e.g., paragraphs[0010], [0052], and [0055]. In some embodiments, the medicamentcomprises a therapeutically effective amount of the at least oneglutarate compound. In some embodiments, the at least one glutaratecompound is a compound of Formula I or Formula II as disclosed herein.In some embodiments, the at least one glutarate compound is an α-KGcompound or a 2-HG compound as disclosed herein. In some embodiments,the at least one glutarate compound is α-ketoglutarate (α-KG),2-hydroxyglutaric acid, and/or (S)-2-hydroxyglutaric acid (S-2HG). Insome embodiments, the medicament comprises a pharmaceutically acceptablecarrier.

In some embodiments, the subject of the methods of the present inventionis a mammalian subject. In some embodiments, the subject is a humansubject. In some embodiments, the subject is one who is in need oftreatment with at least one glutarate compound. As used herein, asubject who is in need of treatment with at least one glutarate compoundis one who has, will, or will likely experience an event that causes anIR injury. In some embodiments, the at least one glutarate compound tobe administered to the subject is exogenous to the subject. In someembodiments, the compositions comprise a concentrated amount of at leastone glutarate compound, wherein the concentrated amount is aconcentration that is higher than naturally occurring concentrations ofthe at least one glutarate compound or its naturally occurringcounterpart as found in nature.

The accompanying drawings are included to provide a furtherunderstanding of the invention and are incorporated in and constitutepart of this specification, illustrate several embodiments of theinvention, and together with the description serve to explain theprinciples of the invention.

DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. This invention is further understood by referenceto the drawings wherein:

FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, and FIG. 6 show thatadministration of α-KG at reperfusion improves heart functional recoveryin ex vivo mouse model. FIG. 1 provides the structure of α-KG andschematically shows the experimental protocol. Isolated, Langendorffhearts from mice were perfused with Krebs Henseleit buffer (KH) andafter stabilization subjected to ischemia for 30 minutes. Afterischemia, hearts were perfused with KH, control (CTRL), or α-KG (800 μM)for 60 minutes. FIG. 2 and FIG. 3 are representative graphs of the leftventricular developed pressure (LVDP) and dP/dt_(Max) and dP/dt_(Min) asa function of time for controls (hearts perfused with KH) (FIG. 2) andhearts perfused with α-KG (FIG. 3). FIG. 4 is a graph showing leftventricular developed pressure (LVDP) as a function of time. FIG. 5 is agraph showing rate pressure product (RPP) as a function of time. FIG. 6is a graph showing the maximum rate of left ventricle (LV) pressure rise(dP/dt_(max)) as a function of time. *p<0.05.

FIG. 7, FIG. 8, and FIG. 9 show that α-KG reduces myocardial infarctsize in IR injury in in vivo mouse model. FIG. 7 schematically shows theexperimental protocol. The LAD was occluded for 45 minutes followed by24 hours of reperfusion. One single IV bolus of phosphate bufferedsaline (PBS) (water as vehicle), α-KG (800 μM final in blood), oroligomycin (10 nM final in blood) was administered at reperfusion. FIG.8 are representative triphenyl tetrazolium chloride (TTC) stainedcross-section of the heart. Infarcted tissue is white, the rest of thearea at risk is red, and non-risk tissue is dark blue. FIG. 9 is a graphshowing the quantification of myocardial infarct size. Data show resultsof 4-5 biological replicates. Bars indicate mean ±SEM. ****P<0.0001,**P=0.0012, NS (not significant), by unpaired t-test, two-tailed,two-sample unequal variance.

FIG. 10 and FIG. 11 show that α-KG inhibits ATP synthase in mouse heartmitochondria. FIG. 10 is a graph showing the inhibition of heart ATPsynthase by α-KG. α-KG decreases state 3 (initiated by 2 mM ADP), butnot state 4o (oligomycin insensitive, that is, complex V independent) or3u (FCCP-uncoupled maximal respiratory capacity), respiration inmitochondria isolated from mouse heart. Oligo, oligomycin; FCCP,carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; AA, antimycin A.In the graph, the high data points in State 3 and State 3u are Vehicle.FIG. 11 is a graph showing that α-KG does not affect the electron flowthrough the ETC. Oxygen consumption rate (OCR) from isolated mouse heartmitochondria at basal (pyruvate and malate as Complex I substrates, inpresence of FCCP) and in response to sequential injection of rotenone(Rote; Complex I inhibitor), succinate (Complex II substrate), antimycinA (AA; complex III inhibitor), tetramethylphenylenediamine (TMPD;cytochrome c (Complex IV) substrate). No difference in Complex I (C I),Complex II (C II), or Complex IV (C IV) respiration is detected. In thegraph, the high data points in State C II are Vehicle.

FIG. 12 are representative triphenyl tetrazolium chloride (TTC) stainedcross-sections of the heart showing protection by α-KG is independent ofHIF and PHD. The LAD was occluded for 45 minutes followed by 24 hours ofreperfusion. One single IV bolus of echinomycin (227 nM), echinomycin(227 nM) plus α-KG (800 μM), or S-2HG (800 μM) was administered atreperfusion. Infarcted tissue is white, the rest of the area at risk isred, and non-risk tissue is dark blue.

FIG. 13 are representative triphenyl tetrazolium chloride (TTC) stainedcross-sections of the heart showing that metabolic conversion of α-KG tosuccinyl-CoA may contribute to protection. The LAD was occluded for 45minutes followed by 24 hours of reperfusion. One single IV bolus of α-KG(800 μM) succinyl-CoA (800 μM) or succinate (800 μM) was administered atreperfusion. Infarcted tissue is white, the rest of the area at risk isred, and non-risk tissue is dark blue.

FIG. 14 is an immunoblot illustrating an increase in cyclophilin Dbinding to Complex V induced by α-KG, which indicates that that α-KB mayconfer cardioprotection by inhibiting the induction of mPTP openingduring reperfusion. Extracts and immunoprecipitates of mouse heartmitochondria using ATP synthase immunoprecipitation kit (Abcam,ab190715) were immunoblotted for ATP5B and CypD. Sample preparation andimmunoprecipitation were performed according to manufacturer's standardprotocols. α-KG and oligomycin were dissolved in water. Bz-423 wasdissolved in DMSO. Water and DMSO were used as respective vehiclecontrols (water for α-KG and oligomycin set of experiments; and DMSO forthe Bz-423 experiments).

DETAILED DESCRIPTION OF THE INVENTION

Ischemia or ischaemia is a restriction in blood supply to tissues,caused by a shortage of oxygen and glucose for cellular metabolism.Reperfusion (or reoxygenation) injury describes tissue damage caused bya return of blood to the tissue after a period of ischemia, anoxia, orhypoxia.

Because the drastic metabolic imbalance incurred by hypoxia, and thatwhich results from the resupply of oxygen and nutrients uponreperfusion, impose severe stresses on the myocardium, the heart wasused as a model for IR injury. Since all IR injuries are the result ofischemic and/or reperfusion conditions, these experiments and resultsusing the heart as a model can be applied to other tissues and organsthat are susceptible to IR injury, including brain, liver, kidney, andlung tissues.

As used herein, the term “ischemia-reperfusion injury” (IR injury)refers to tissue damage caused by ischemia, reperfusion, or ischemiafollowed by reperfusion. Thus, the term “IR injury” includes injuriescaused by ischemia, reperfusion injuries, and injuries caused byischemia followed by reperfusion. As used herein, an “event that causesan IR injury” includes ischemia, reperfusion, thromboembolism,vasoconstriction, cardiac standstill, myocardial infarction, stroke,surgeries in which blood flow to a tissue is prevented or reduced (e.g.,organ transplantations, heart surgery, etc.), and the like. Myocardialinfarction (MI) is a type of cardiac ischemia that can result in IRinjury of the heart tissues.

Endogenous metabolites (including normal as well as aberrantdisease-associated metabolites) were screened for their potentialeffects against heart IR injury using ex vivo and in vivo mouse models.From the screen, the tricarboxylic acid (TCA) cycle intermediateα-ketoglutarate (α-KG) was found to confer the greatest protection. Infact, one single bolus of α-KG at the onset of reperfusion reduced theinfarct size in mice by about 70%. As disclosed herein, α-KG is apowerful therapeutic agent that can be used to rapidly restore heartfunction and reduce infarct size resulting from ischemia and/orreperfusion, caused by, for example, myocardial infarction. Thus, thepresent invention is directed to preventing, inhibiting, reducing, ortreating IR injury to a tissue in a subject, which comprisesadministering to the subject a therapeutically effective amount of aglutarate compound, such as α-KG.

As used herein, a “glutarate compound” refers to α-KG compounds, 2-HGcompounds, and compounds having the following Formula I:

wherein

R¹ is hydrogen, halogen, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —SR¹⁰,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl;

R² and R³ are each independently hydrogen, halogen, —CN, —CHO, —OR⁷,—NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted or unsubstitutedalkyl, or substituted or unsubstituted heteroalkyl; or R² and R³,together with the atom to which they are bound, form an oxo; and

R⁴, R⁵, and R⁶ are each independently hydrogen, halogen, —CN, —CHO,—OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl,wherein R⁷, R⁸, R⁹, and R¹⁰ are each independently hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl,

and pharmaceutically acceptable solvates, salts, prodrugs, andmetabolites thereof.

In some embodiments, R¹ is hydrogen, halogen, —CHO, —OR⁷, —NR⁸R⁹,—COOR⁷, —CONR⁸R⁹, or —SR¹⁰. In some embodiments, R¹ is hydrogen, —CHO,or —OR⁷. In some embodiments, R¹ is —OR⁷, wherein R⁷ is hydrogen,substituted or unsubstituted alkyl, or substituted or unsubstitutedaryl. In some embodiments, R¹ is —OR⁷, wherein R⁷ is C₁₋₂₀ substitutedor unsubstituted alkyl.

In some embodiments, R² and R³ are each independently hydrogen, halogen,—CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl. Insome embodiments, R² and R³ are each independently hydrogen, halogen,—CN, —CHO, or —NR⁸R⁹, wherein R⁸ and R⁹ are each independently hydrogenor substituted or unsubstituted alkyl. In some embodiments, R² and R³,together with the atom to which they are bound, form an oxo.

In some embodiments, R⁴, R⁵, and R⁶ are each independently hydrogen,—CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹. In some embodiments, R⁴, R⁵,and R⁶ are each independently hydrogen, halogen, —OR⁷, —NR⁸R⁹, —COOR⁷,—CONR⁸R⁹, or substituted or unsubstituted alkyl. In some embodiments, R⁴and R⁵ are each independently hydrogen, halogen, —CN, —CHO, —OR⁷,—NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted or unsubstitutedalkyl, or substituted or unsubstituted heteroalkyl; and R⁶ is halogen,—CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl. Insome embodiments, R⁴ and R⁵ are each independently hydrogen, —CHO, —OR⁷,—NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹; and R⁶ is —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, or—CONR⁸R⁹.

In some embodiments, R¹ is —OR⁷ or —NR⁸R⁹; R² and R³ are eachindependently hydrogen, —CHO, —OR⁷, —NR⁸R⁹, or unsubstituted alkyl, orR² and R³, together with the atom to which they are bound, form an oxo;R⁴ and R⁵ are each independently hydrogen, —OR⁷, —NR⁸R⁹, orunsubstituted alkyl; R⁶ is —NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹, and R⁷, R⁸, R⁹,and R¹⁰ are each independently hydrogen, or substituted or unsubstitutedalkyl.

In some embodiments, R¹ is —OR⁷ or —NR⁸R⁹; R² and R³ are eachindependently hydrogen, —CHO, —OR⁷, —NR⁸R⁹, or unsubstituted alkyl, orR² and R³, together with the atom to which they are bound, form an oxo;R⁴, R⁵, and R⁶ are each independently hydrogen, —OR⁷, —NR⁸R⁹, orunsubstituted alkyl; and R⁷, R⁸, R⁹, and R¹⁰ are each independentlyhydrogen, or substituted or unsubstituted alkyl.

In some embodiments, R¹ is —OR⁷; R² and R³, together with the atom towhich they are bound, form an oxo; R⁴ and R⁵ are each independentlyhydrogen or unsubstituted alkyl; R⁶ is —COOR⁷, R⁷ is hydrogen; and R⁸,R⁹, and R¹⁰ are each independently hydrogen or unsubstituted alkyl.

In some embodiments, R⁷, R⁸, R⁹, and R¹⁰ are each independently hydrogenor C₁₋₂₀ substituted or unsubstituted alkyl. In some embodiments, R⁷,R⁸, R⁹, and R¹⁰ are each independently hydrogen or C₁₋₁₀ substituted orunsubstituted alkyl. In some embodiments, R⁷, R⁸, R⁹, and R¹⁰ are eachindependently hydrogen or C₁₋₅ substituted or unsubstituted alkyl.

In some embodiments, the glutarate compound of Formula I has thefollowing Formula II:

wherein

Ra and Rb are each independently a negative charge, hydrogen, Na, astraight or branched C₁-C₁₀ alkyl, or a straight or branched C₁-C₁₀alkenyl, and

Rc is optionally present, and if present, Rc is hydrogen, a straight orbranched C₁-C₁₀ alkyl, or a straight or branched C₁-C₁₀ alkenyl, and ifabsent, Z is a double bond,

and pharmaceutically acceptable solvates, salts, prodrugs, andmetabolites thereof.

In some embodiments, Ra and Rb are each independently a negative charge,hydrogen, Na, a straight or branched C₁-C₅ alkyl, or a straight orbranched C₁-C₅ alkenyl.

In some embodiments, if present, Rc is hydrogen, a straight or branchedC₁-C₅ alkyl, or a straight or branched C₁-C₅ alkenyl.

In some embodiments, the glutarate compound is:

or a salt thereof. In some embodiments, the glutarate compound is:

or a salt thereof.

As used herein, an “α-KG compound” refers to α-ketoglutarate (α-KG),derivatives of α-ketoglutarate (e.g., the derivatives set forth inMacKenzie, et al. (2007) Mol Cell Biol 27 (9):3282-3289)), analogues ofα-ketoglutarate (e.g., phosphonate analogues (e.g., those recited inBunik, et al. (2005) Biochemistry 44 (31):10552-61), esters ofα-ketoglutarate (e.g., dimethyl α-ketoglutarate and octylα-ketoglutarate), and various species specific analogues, e.g., humanα-ketoglutarate, porcine α-ketoglutarate, murine α-ketoglutarate, bovineα-ketoglutarate, and the like. In some embodiments, the α-KG compound isα-ketoglutaric acid. In some embodiments, the α-KG compound is dimethylα-ketoglutarate. In some embodiments, the α-KG compound is octylα-ketoglutarate. As used herein, “α-ketoglutarate” and “α-ketoglutaricacid” are used interchangeably. As used herein, the abbreviation “KG”may be used to refer to the term “ketoglutarate”, e.g., α-ketoglutarateis abbreviated as α-KG.

As used herein, a “2-HG compound” refers to 2-hydroxyglutaric acid,2-hydroxypentanedioate, and compounds having 2-hydroxypentanedioate aspart of its backbone structure and includes1-alkyl-(S)-2-hydroxypentanedioate, 1-alkyl-(R)-2-hydroxypentanedioate,1-alkenyl-(S)-2-hydroxypentanedioate,1-alkenyl-(R)-2-hydroxypentanedioate,5-alkyl-(S)-2-hydroxypentanedioate, 5-alkyl-(R)-2-hydroxypentanedioate,5-alkenyl-(S)-2-hydroxypentanedioate, and5-alkenyl-(R)-2-hydroxypentanedioate, wherein alkyl is a straight orbranched C₁-C₁₀ alkyl and alkenyl is a straight or branched C₁-C₁₀alkenyl. In some embodiments, the 2-HG compound is1-octyl-(S)-2-hydroxypentanedioate, 1-octyl-(R)-2-hydroxypentanedioate,5-octyl-(S)-2-hydroxypentanedioate, or5-octyl-(R)-2-hydroxypentanedioate. In some embodiments, the 2-HGcompound is disodium (S)-2-hydroxyglutarate or (S)-2-hydroxyglutaricacid (S-2HG). In some embodiments, the 2-HG compound isL-α-hydroxyglutaric acid disodium salt. As used herein, the abbreviation“HG” may be used to refer to the term “hydroxypentanedioate”, e.g.,2-hydroxypentanedioate is abbreviated as 2-HG. As used herein,“2-hydroxyglutarate” and “2-hydroxyglutaric acid” are usedinterchangeably. In some embodiments, a 2-HG compound is S-2HG.

A “pharmaceutically acceptable solvate” refers to a solvate form of aspecified compound that retains the biological effectiveness of thespecified compound. Examples of solvates include compounds of theinvention in combination with water, isopropanol, ethanol, methanol,dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine, oracetone. Those skilled in the art of organic chemistry will appreciatethat many organic compounds can form complexes with solvents in whichthey are reacted or from which they are precipitated or crystallized.These complexes are known as “solvates”. For example, a complex withwater is known as a “hydrate”. Solvates of compounds of glutaratecompounds are within the scope of the invention. It will also beappreciated by those skilled in organic chemistry that many organiccompounds can exist in more than one crystalline form. For example,crystalline form may vary from solvate to solvate. Thus, all crystallineforms of the glutarate compounds or the pharmaceutically acceptablesolvates thereof are within the scope of the present invention.

The term “pharmaceutically acceptable salt” refers to a salt form thatis pharmacologically acceptable and substantially non-toxic to thesubject being treated with the compound of the invention.pharmaceutically acceptable salts include conventional acid-additionsalts or base-addition salts formed from suitable non-toxic organic orinorganic acids or inorganic bases. Exemplary acid-addition saltsinclude those derived from inorganic acids such as hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid,phosphoric acid, and nitric acid, and those derived from organic acidssuch as p-toluenesulfonic acid, methanesulfonic acid, ethane-disulfonicacid, isethionic acid, oxalic acid, p-bromophenylsulfonic acid, carbonicacid, succinic acid, citric acid, benzoic acid, 2-acetoxybenzoic acid,acetic acid, phenylacetic acid, propionic acid, glycolic acid, stearicacid, lactic acid, malic acid, tartaric acid, ascorbic acid, maleicacid, hydroxymaleic acid, glutamic acid, salicylic acid, sulfanilicacid, and fumaric acid. Exemplary base-addition salts include thosederived from ammonium hydroxides (e.g., a quaternary ammonium hydroxidesuch as tetramethylammonium hydroxide), those derived from inorganicbases such as alkali or alkaline earth-metal (e.g., sodium, potassium,lithium, calcium, or magnesium) hydroxides, and those derived fromnon-toxic organic bases such as basic amino acids.

A “pharmaceutically acceptable prodrug” is a compound that may beconverted under physiological conditions or by solvolysis to thespecified compound or to a pharmaceutically acceptable salt of suchcompound. A “pharmaceutically active metabolite” refers to apharmacologically active product produced through metabolism in the bodyof a specified compound or salt thereof. Prodrugs and active metabolitesof a compound may be identified using routine techniques known in theart. See, e.g., Bertolini, G. et al., (1997) J. Med. Chem. 40:2011-2016;Shan, D. et al., J. Pharm. Sci., 86 (7):765-767; Bagshawe K., (1995)Drug Dev. Res. 34:220-230; Bodor, N., (1984) Advances in Drug Res.13:224-331; Bundgaard, H., Design of Prodrugs (Elsevier Press, 1985) andLarsen, I. K., Design and Application of Prodrugs, Drug Design andDevelopment (Krogsgaard-Larsen et al., eds., Harwood AcademicPublishers, 1991).

Compositions of the present invention, including pharmaceuticalcompositions, may include one glutarate compound or two or moredifferent glutarate compounds, e.g., an α-KG compound and a 2-HGcompound. As used herein, the terms “pharmaceutical composition” and“pharmaceutical formulation” are used interchangeably to refer to acomposition suitable for pharmaceutical use in a subject. Apharmaceutical composition generally comprises an effective amount of anactive agent, e.g., a glutarate compound and a pharmaceuticallyacceptable carrier, e.g., a buffer, adjuvant, diluent, and the like. Insome embodiments, the compositions, including pharmaceuticalcompositions comprise a concentrated amount of at least one glutaratecompound, wherein the concentrated amount is a concentration that ishigher than naturally occurring concentrations of the at least oneglutarate compound or its naturally occurring counterpart as found innature.

As used herein, “pharmaceutically acceptable vehicle” and“pharmaceutically acceptable carrier” are used interchangeably and referto and include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, excipients, and the like, that are compatible withpharmaceutical administration and comply with applicable standards andregulations, e.g., the pharmacopeial standards set forth in the UnitedStates Pharmacopeia and the National Formulary (USP-NF) book, forpharmaceutical administration. Thus, for example, unsterile water isexcluded as a pharmaceutically acceptable carrier for, at least,intravenous administration. pharmaceutically acceptable vehicles includethose known in the art. See, e.g., Remington: The Science and Practiceof Pharmacy. 20^(th) ed. (2000) Lippincott Williams & Wilkins.Baltimore, Md., which is herein incorporated by reference.

One or more glutarate compounds according to the present invention maybe administered, preferably in the form of pharmaceutical compositions,to a subject. Preferably the subject is mammalian, more preferably, thesubject is human. Preferred pharmaceutical compositions are thosecomprising at least one glutarate compound in a therapeuticallyeffective amount, and a pharmaceutically acceptable vehicle.

In some embodiments, the amount of the one or more glutarate compoundsadministered to the subject is a therapeutically effective amount or aneffective amount. As used herein, an “effective amount” is a dose thatresults in an observable difference as compared to a placebo. A“therapeutically effective amount”, refers to an amount of one or morecompounds of the present invention that, when administered to a subject,(i) treats or inhibits the particular disease, condition, or disorder,(ii) attenuates, ameliorates, or eliminates one or more symptoms of theparticular disease, condition, or disorder, and/or (iii) inhibits ordelays the onset of one or more symptoms of the particular disease,condition, or disorder, as compared to a control. A therapeuticallyeffective amount of one or more compounds of the present invention willvary depending upon factors such as the given compound(s), thepharmaceutical formulation, route of administration, the type of diseaseor disorder, the degree of the disease or disorder, and the identity ofthe subject being treated, but can nevertheless be readily determined byone skilled in the art. For example, a “therapeutically effectiveamount” of a glutarate compound is one that inhibits or reduces theamount of an IR injury as compared to a negative control.

In some embodiments, one or more glutarate compounds are administeredprior to, during, and/or after an event that causes an IR injury or aperiod of ischemia and/or reperfusion. In some embodiments, atherapeutically effective amount one or more glutarate compounds isadministered prior to, during, and/or after the event or the period ofischemia and/or reperfusion. In some embodiments, the event ismyocardial infarction, stroke, surgeries in which blood flow to a tissueis prevented, inhibited, or reduced (such as in organ transplantations),and the like. In some embodiments, the event is myocardial infarction.In some embodiments, the administration is immediately prior to theevent or the period of ischemia and/or reperfusion. In some embodiments,the administration is up to 2 hours, preferably up to 1 hour, after theevent or the period of ischemia and/or reperfusion. In some embodiments,the administration is within 30 minutes of the event or the period ofischemia and/or reperfusion. In some embodiments, the administration iswithin 1 to 2 hours of the event. In some embodiments, theadministration is within 1 hour of the event. In some embodiments, theadministration is within 30 minutes, 25 minutes, 20 minutes, 15 minutes,10 minutes, or 5 minutes of the event. In some embodiments, theadministration is within 30 minutes of the event. In some embodiments,the administration is within 25 minutes of the event. In someembodiments, the administration is within 20 minutes of the event. Insome embodiments, the administration is within 15 minutes of the event.In some embodiments, the administration is within 10 minutes of theevent. In some embodiments, the administration is within 5 minutes ofthe event. In some embodiments, the administration is within 1 minute ofthe event. In some embodiments, the administration is immediately afterthe occurrence of the event.

In some embodiments, a therapeutically effective amount of the one ormore glutarate compounds are administered as a single dose of about5-20, about 10-15, or about 11-12 milligrams per kilogram weight of thesubject prior to, during, or after the event that causes an IR injury.In some embodiments, a therapeutically effective amount of at least oneα-KG compound, such as α-KG, is administered as a single dose of about5-15, about 10-15, or about 11-12 milligrams per kilogram weight of thesubject prior to, during, or after the event that causes an IR injury.In some embodiments, the at least one glutarate compound is α-KG and thetherapeutically effective amount is about 11.5 milligrams per kilogramweight of the subject. In some embodiments, a therapeutically effectiveamount of at least one 2-HG compound, such as S-2HG, is administered asa single dose of about 10-20, about 15-20, or about 15-16 milligrams perkilogram weight of the subject prior to, during, or after the event thatcauses an IR injury. In some embodiments, the at least one glutaratecompound is S-2HG and the therapeutically effective amount is about 15.5milligrams per kilogram weight of the subject. In some embodiments, thetherapeutically effective amount is about 5-50 mg/kg, 5-40 mg/kg, 5-30mg/kg, 5-20 mg/kg, 5-10 mg/kg, 10-40 mg/kg, 10-30 mg/kg, 10-20 mg/kg, orabout 10-15 mg/kg of the subject. In some embodiments, thetherapeutically effective amount is about 5-20 mg/kg, or about 10-15mg/kg of the subject. In some embodiments, the therapeutically effectiveamount is about 5-50 mg/kg. In some embodiments, the therapeuticallyeffective amount is about 5-40 mg/kg. In some embodiments, thetherapeutically effective amount is about 5-30 mg/kg. In someembodiments, the therapeutically effective amount is about 5-20 mg/kg.In some embodiments, the therapeutically effective amount is about 5-10mg/kg. In some embodiments, the therapeutically effective amount isabout 10-40 mg/kg. In some embodiments, the therapeutically effectiveamount is about 10-30 mg/kg. In some embodiments, the therapeuticallyeffective amount is about 10-20 mg/kg. In some embodiments, thetherapeutically effective amount is about 10-15 mg/kg. In someembodiments, the timing of administration of the therapeuticallyeffective amount of one or more glutarate compounds is in accordancewith paragraph [0051]. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present.

The one or more glutarate compounds to be administered to a subject maybe provided as a pharmaceutical formulation. pharmaceutical formulationsmay be prepared in a unit-dosage form appropriate for the desired modeof administration. The pharmaceutical formulations of the presentinvention may be administered by any suitable route including oral,rectal, nasal, topical (including buccal and sublingual), vaginal, andparenteral (including subcutaneous, intramuscular, intravenous, andintradermal). It will be appreciated that the route of administrationmay vary with the condition and age of the recipient, the nature of thecondition to be treated, and the given compound(s) of the presentinvention. In some embodiments, the route of administration is oral. Insome embodiments, the pharmaceutical composition is formulated forparenteral administration. In some embodiments, the pharmaceuticalcomposition is formulated for subcutaneous administration. In someembodiments, the pharmaceutical composition is formulated forintramuscular administration. In some embodiments, the pharmaceuticalcomposition is formulated for intravenous administration. In someembodiments, the pharmaceutical composition is formulated forintraarterial administration. In some embodiments, the pharmaceuticalcomposition is formulated for intradermal administration.

It will be appreciated that the actual dosages of the glutaratecompounds used in the pharmaceutical formulations will vary according tothe particular compound(s) being used, the particular compositionformulated, the mode of administration, and the particular site,subject, and disease being treated. Optimal dosages for a given set ofconditions may be ascertained by those skilled in the art using dosagedetermination tests in view of the experimental data for a givencompound. Administration of prodrugs may be dosed at weight levels thatare chemically equivalent to the weight levels of the fully activeforms.

In some embodiments, a therapeutically effective amount of one or moreglutarate compounds is administered as a daily dose of about 0.01-2,about 0.25-2, about 0.5-2, about 1-2, or about 2 grams per kilogramweight of the subject per day. In some embodiments, a therapeuticallyeffective amount of one or more glutarate compounds is administered as adaily dose of about 0.1-1, about 0.25-1, about 0.5-1, or about 1 gramper kilogram weight of the subject per day. In some embodiments, one ormore glutarate compounds is administered as a daily dose of about0.01-1.0, about 0.01-0.5, or about 0.1-0.2 grams per kilogram weight ofthe subject per day. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present.

The therapeutically effective amount may be administered as a singledose or as multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or moredoses) over a period of time. For example, a subject may be treated withone or more glutarate compounds at least once. Alternatively, thesubject may be treated with one or more compounds of glutarate compoundsfrom about one time per week to about once daily for a given treatmentperiod. The length of the treatment period will depend on a variety offactors such as the severity of the disease or disorder, theconcentration and activity of the one or more compounds of the presentinvention, or a combination thereof. It will also be appreciated thatthe effective dosage of the one or more glutarate compounds may increaseor decrease over the course of a particular treatment.

In some embodiments, one or more glutarate compounds are administered toa subject in combination with an additional therapeutic agent. In someembodiments, the additional therapeutic agent is oligomycin. In someembodiments, the additional therapeutic agent is aspirin.

Toxicity and therapeutic efficacy of the one or more glutarate compoundscan be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. Compounds exhibiting large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

In some embodiments, the present invention is directed to kits whichcomprise one or more glutarate compounds, optionally in a composition orin combination with one or more additional therapeutic agents, packagedtogether with one or more reagents or drug delivery devices forpreventing, inhibiting, reducing, or treating an IR injury. Such kitsinclude a carrier, package, or container that may be compartmentalizedto receive one or more containers, such as vials, tubes, and the like.In some embodiments, the kits optionally include an identifyingdescription or label or instructions relating to its use. In someembodiments, the kits comprise the one or more glutarate compounds,optionally in one or more unit dosage forms, packaged together as a packand/or in drug delivery device, e.g., a pre-filled syringe. In someembodiments, the kits include information prescribed by a governmentalagency that regulates the manufacture, use, or sale of compounds andcompositions according to the present invention.

Additional Exemplary Methods of Use

The present invention provides a method of treating or reducing anischemia-reperfusion injury (IR injury) or a method of preventing orinhibiting an ischemia-reperfusion injury (IR injury) in a subject inneed thereof, comprising administering to the subject a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula I:

wherein: R¹ is hydrogen, halogen, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹,—SR¹⁰, substituted or unsubstituted alkyl, or substituted orunsubstituted heteroalkyl; R² and R³ are each independently hydrogen,halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl; or R² and R³, together with the atom to which they arebound, form an oxo; R⁴, R⁵, and R⁶ are each independently hydrogen,halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl; and R⁷, R⁸, R⁹, and R¹⁰ are each independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl, or a salt thereof; wherein thepharmaceutical composition is administered to the subject to treat,reduce, prevent, or inhibit IR injury to a tissue in the subject. Apharmaceutical composition described herein may further comprise anexcipient.

In some embodiments of a compound of Formula I, R¹ is hydrogen, halogen,—CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, or —SR¹⁰. In some embodiments, R¹is hydrogen, —CHO, or —OR⁷. In some embodiments, R¹ is —OR⁷, wherein R⁷is hydrogen, substituted or unsubstituted alkyl, or substituted orunsubstituted aryl. In some embodiments, R¹ is —OR⁷, wherein R⁷ is C₁₋₂₀substituted or unsubstituted alkyl.

In some embodiments of a compound of Formula I, R² is hydrogen, halogen,—CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl and R³is hydrogen, halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂,—SR¹⁰, substituted or unsubstituted alkyl, or substituted orunsubstituted heteroalkyl. In some embodiments, R² is hydrogen, halogen,—CN, —CHO, or —NR⁸R⁹, wherein R⁸ and R⁹ are each independently hydrogenor substituted or unsubstituted alkyl. In some embodiments, R³ ishydrogen, halogen, —CN, —CHO, or —NR⁸R⁹, wherein R⁸ and R⁹ are eachindependently hydrogen or substituted or unsubstituted alkyl. In someembodiments, R² and R³, together with the atom to which they are bound,form an oxo.

In some embodiments of a compound of Formula I, R⁴ is hydrogen, —CHO,—OR⁷, —NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹. In some embodiments, R⁵ is hydrogen,—CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹. In some embodiments, R⁶ ishydrogen, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹.

In some embodiments of a compound of Formula I, R⁷ is hydrogen or C₁₋₂₀substituted or unsubstituted alkyl. In some embodiments, R⁸ is hydrogenor C₁₋₂₀ substituted or unsubstituted alkyl. In some embodiments, R⁹ ishydrogen or C₁₋₂₀ substituted or unsubstituted alkyl.

In some embodiments, the present invention provides a method of treatingor reducing an ischemia-reperfusion injury (IR injury) or a method ofpreventing or inhibiting an ischemia-reperfusion injury (IR injury) in asubject in need thereof, comprising administering to the subject apharmaceutical composition comprising a therapeutically effective amountof a compound of Formula I:

wherein: R¹ is —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —SR¹⁰, R² and R³are each independently hydrogen, halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹,—SR¹⁰, or substituted or unsubstituted alkyl; or R² and R³, togetherwith the atom to which they are bound, form an oxo; R⁴, R⁵, and R⁶ areeach independently hydrogen, halogen, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, orsubstituted or unsubstituted alkyl; and R⁷, R⁸, R⁹, and R¹⁰ are eachindependently hydrogen, substituted or unsubstituted alkyl, orsubstituted or unsubstituted heteroalkyl; or a salt thereof.

In some embodiments, the present invention provides a method of treatingor reducing an ischemia-reperfusion injury (IR injury) or a method ofpreventing or inhibiting an ischemia-reperfusion injury (IR injury) in asubject in need thereof, comprising administering to the subject apharmaceutical composition comprising a therapeutically effective amountof a compound of Formula I:

wherein: R¹ is —OR⁷ or —NR⁸R⁹; R² and R³ are each independentlyhydrogen, —CHO, —OR⁷, —NR⁸R⁹, or unsubstituted alkyl; or R² and R³,together with the atom to which they are bound, form an oxo; R⁴, R⁵, andR⁶ are each independently hydrogen, —OR⁷, —NR⁸R⁹, or unsubstitutedalkyl; and R⁷, R⁸, R⁹, and R¹⁰ are each independently hydrogen, orsubstituted or unsubstituted alkyl; or a salt thereof.

In some embodiments, the present invention provides a method of treatingor reducing an ischemia-reperfusion injury (IR injury) or a method ofpreventing or inhibiting an ischemia-reperfusion injury (IR injury) in asubject in need thereof, comprising administering to the subject apharmaceutical composition comprising a therapeutically effective amountof a compound of Formula I:

wherein: R¹ is —OR⁷; R² and R³, together with the atom to which they arebound, form an oxo; R⁴, R⁵, and R⁶ are each independently hydrogen orunsubstituted alkyl; and R⁷, R⁸, R⁹, and R¹⁰ are each independentlyhydrogen or unsubstituted alkyl; or a salt thereof.

The present invention provides a method of treating or reducing anischemia-reperfusion injury (IR injury) to a tissue in a subject,comprising administering to the subject a therapeutically effectiveamount of a pharmaceutical composition comprising: a therapeuticallyeffective amount of a compound of Formula III:

wherein: R¹ is hydrogen, halogen, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹,—SR¹⁰, substituted or unsubstituted alkyl, or substituted orunsubstituted heteroalkyl; R² and R³ are each independently hydrogen,halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰ ,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl; or R² and R³, together with the atom to which they arebound, form an oxo; R⁴ and R⁵ are each independently hydrogen, halogen,—CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R⁶ ishalogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl; and R⁷, R⁸, R⁹, and R¹⁰ are each independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; or a salt thereof; wherein thepharmaceutical composition is administered to the subject to treat orreduce IR injury to a tissue in the subject.

In some embodiments of a compound of Formula III, R¹ is hydrogen,halogen, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, or —SR¹⁰. In someembodiments, R¹ is hydrogen, —CHO, or —OR⁷. In some embodiments, R¹ is—OR⁷, wherein R⁷ is hydrogen, substituted or unsubstituted alkyl, orsubstituted or unsubstituted aryl. In some embodiments, R¹ is —OR⁷,wherein R⁷ is C₁₋₂₀ substituted or unsubstituted alkyl.

In some embodiments of a compound of Formula III, R² is hydrogen,halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl and R³ is hydrogen, halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹,—COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted or unsubstituted alkyl, orsubstituted or unsubstituted heteroalkyl. In some embodiments, R² ishydrogen, halogen, —CN, —CHO, or —NR⁸R⁹, wherein R⁸ and R⁹ are eachindependently hydrogen or substituted or unsubstituted alkyl. In someembodiments, R³ is hydrogen, halogen, —CN, —CHO, or —NR⁸R⁹, wherein R⁸and R⁹ are each independently hydrogen or substituted or unsubstitutedalkyl. In some embodiments, R² and R³, together with the atom to whichthey are bound, form an oxo.

In some embodiments of a compound of Formula III, R⁴ is hydrogen, —CHO,—OR⁷, —NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹. In some embodiments, R⁵ is hydrogen,—CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹. In some embodiments, R⁶ is—CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹.

In some embodiments of a compound of Formula III, R⁷ is hydrogen orC₁₋₂₀ substituted or unsubstituted alkyl. In some embodiments, R⁸ ishydrogen or C₁₋₂₀ substituted or unsubstituted alkyl. In someembodiments, R⁹ is hydrogen or C₁₋₂₀ substituted or unsubstituted alkyl.

In some embodiments, the present invention provides a method of treatingor reducing an ischemia-reperfusion injury (IR injury) or a method ofpreventing or inhibiting an ischemia-reperfusion injury (IR injury) in asubject in need thereof, comprising administering to the subject apharmaceutical composition comprising a therapeutically effective amountof a compound of Formula III:

wherein: R¹ is —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —SR¹⁰, R² and R³are each independently hydrogen, halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹,—SR¹⁰, or substituted or unsubstituted alkyl; or R² and R³, togetherwith the atom to which they are bound, form an oxo; R⁴ and R⁵ are eachindependently hydrogen, halogen, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, orsubstituted or unsubstituted alkyl; R⁶ is halogen, —CN, —CHO, —OR⁷,—NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted or unsubstitutedalkyl, or substituted or unsubstituted heteroalkyl; and R⁷, R⁸, R⁹, andR¹⁰ are each independently hydrogen, substituted or unsubstituted alkyl,or substituted or unsubstituted heteroalkyl; or a salt thereof.

In some embodiments, the present invention provides a method of treatingor reducing an ischemia-reperfusion injury (IR injury) or a method ofpreventing or inhibiting an ischemia-reperfusion injury (IR injury) in asubject in need thereof, comprising administering to the subject apharmaceutical composition comprising a therapeutically effective amountof a compound of Formula III:

wherein: R¹ is —OR⁷ or —NR⁸R⁹; R² and R³ are each independentlyhydrogen, —CHO, —OR⁷, —NR⁸R⁹,or unsubstituted alkyl; or R² and R³,together with the atom to which they are bound, form an oxo; R⁴ and R⁵are each independently hydrogen, —OR⁷, —NR⁸R⁹, or unsubstituted alkyl;R⁶ is —NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹, and R⁷, R⁸, R⁹, and R¹⁰ are eachindependently hydrogen, or substituted or unsubstituted alkyl; or a saltthereof.

In some embodiments, the present invention provides a method of treatingor reducing an ischemia-reperfusion injury (IR injury) or a method ofpreventing or inhibiting an ischemia-reperfusion injury (IR injury) in asubject in need thereof, comprising administering to the subject apharmaceutical composition comprising a therapeutically effective amountof a compound of Formula III:

wherein: R¹ is —OR⁷; R² and R³, together with the atom to which they arebound, form an oxo; R⁴ and R⁵ are each independently hydrogen orunsubstituted alkyl; R⁶ is —COOR⁷, R⁷ is hydrogen; and R⁸, R⁹, and R¹⁰are each independently hydrogen or unsubstituted alkyl; or a saltthereof.

In some embodiments, the present invention provides a method of treatingor reducing an ischemia-reperfusion injury (IR injury) or a method ofpreventing or inhibiting an ischemia-reperfusion injury (IR injury) in asubject in need thereof, comprising administering to the subject apharmaceutical composition comprising a therapeutically effective amountof a compound represented by the structure:

or a salt thereof.

In some embodiments, the present invention provides a method of treatingor reducing an ischemia-reperfusion injury (IR injury) or a method ofpreventing or inhibiting an ischemia-reperfusion injury (IR injury) in asubject in need thereof, comprising administering to the subject apharmaceutical composition comprising a therapeutically effective amountof a compound represented by the structure:

or a salt thereof.

In some embodiments, a compound of Formula I or Formula III describedabove is administered during an event that causes IR injury, reperfusioninjury, or both. In some embodiments, the event is myocardialinfarction, stroke, surgeries in which blood flow to a tissue isprevented such as in organ transplantations, and the like. In someembodiments, the event is myocardial infarction. In some embodiments,the compound of Formula I or Formula III is α-KG. In some embodiments,the compound of Formula I or Formula III is a 2-HG compound. In someembodiments, the compound of Formula I or Formula III is S-2HG.

In other embodiments, a compound of Formula I or Formula III isadministered after an event that causes IR injury, reperfusion injury,or both. In some embodiments, the event is myocardial infarction,stroke, surgeries in which blood flow to a tissue is prevented such asin organ transplantations, and the like. In some embodiments, the eventis myocardial infarction. In some embodiments, the compound of Formula Ior Formula III is α-KG. In some embodiments, the compound of Formula Ior Formula III is a 2-HG compound. In some embodiments, the compound ofFormula I or Formula III is S-2HG.

In some embodiments, a compound of Formula I or Formula III isadministered within 1 to 2 hours of the event. In some embodiments, thecompound of Formula I or Formula III is administered within 1 hour ofthe event. In some embodiments, the compound of Formula I or Formula IIIis administered within 30 minutes, 25 minutes, 20 minutes, 15 minutes,10 minutes, or 5 minutes of the event. In some embodiments, the compoundof Formula I or Formula III is administered within 30 minutes of theevent. In some embodiments, the compound of Formula I or Formula III isadministered within 25 minutes of the event. In some embodiments, thecompound of Formula I or Formula III is administered within 20 minutesof the event. In some embodiments, the compound of Formula I or FormulaIII is administered within 15 minutes of the event. In some embodiments,the compound of Formula I or Formula III is administered within 10minutes of the event. In some embodiments, the compound of Formula I orFormula III is administered within 5 minutes of the event. In someembodiments, the compound of Formula I or Formula III is administeredwithin 1 minute of the event. In some embodiments, the compound ofFormula I or Formula III is administered immediately after theoccurrence of the event. In some embodiments, the event is myocardialinfarction, stroke, surgeries in which blood flow to a tissue isprevented such as in organ transplantations, and the like. In someembodiments, the event is myocardial infarction. In some embodiments,the compound of Formula I or Formula III is α-KG. In some embodiments,the compound of Formula I or Formula III is a 2-HG compound. In someembodiments, the compound of Formula I or Formula III is S-2HG.

In some embodiments, the compound of Formula I or Formula III is α-KG.In some embodiments, α-KG is administered within 1 to 2 hours of theevent. In some embodiments, α-KG is administered within 1 hour of theevent. In some embodiments, α-KG is administered within 30 minutes, 25minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes of the event.In some embodiments, α-KG is administered within 30 minutes of theevent. In some embodiments, α-KG is administered within 25 minutes ofthe event. In some embodiments, α-KG is administered within 20 minutesof the event. In some embodiments, α-KG is administered within 15minutes of the event. In some embodiments, α-KG is administered within10 minutes of the event. In some embodiments, α-KG is administeredwithin 5 minutes of the event. In some embodiments, α-KG is administeredwithin 1 minute of the event. In some embodiments, α-KG is administeredimmediately after the occurance of the event. In some embodiments, theevent is myocardial infarction, stroke, surgeries in which blood flow toa tissue is prevented such as in organ transplantations, and the like.In some embodiments, the event is myocardial infarction.

In some embodiments, the compound of Formula I or Formula III is a 2-HGcompound. In some embodiments, a 2-HG compound is administered within 1to 2 hours of the event. In some embodiments, the 2-HG compound isadministered within 1 hour of the event. In some embodiments, the 2-HGcompound is administered within 30 minutes, 25 minutes, 20 minutes, 15minutes, 10 minutes, or 5 minutes of the event. In some embodiments, the2-HG compound is administered within 30 minutes of the event. In someembodiments, the 2-HG compound is administered within 25 minutes of theevent. In some embodiments, the 2-HG compound is administered within 20minutes of the event. In some embodiments, the 2-HG compound isadministered within 15 minutes of the event. In some embodiments, the2-HG compound is administered within 10 minutes of the event. In someembodiments, the 2-HG compound is administered within 5 minutes of theevent. In some embodiments, the 2-HG compound is administered within 1minute of the event. In some embodiments, the 2-HG compound isadministered immediately after the occurrence of the event. In someembodiments, the event is myocardial infarction, stroke, surgeries inwhich blood flow to a tissue is prevented such as in organtransplantations, and the like. In some embodiments, the event ismyocardial infarction.

In some embodiments, the compound of Formula I or Formula III is S-2HG.In some embodiments, S-2HG is administered within 1 to 2 hours of theevent. In some embodiments, S-2HG is administered within 1 hour of theevent. In some embodiments, S-2HG is administered within 30 minutes, 25minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes of the event.In some embodiments, S-2HG is administered within 30 minutes of theevent. In some embodiments, S-2HG is administered within 25 minutes ofthe event. In some embodiments, S-2HG is administered within 20 minutesof the event. In some embodiments, S-2HG is administered within 15minutes of the event. In some embodiments, S-2HG is administered within10 minutes of the event. In some embodiments, S-2HG is administeredwithin 5 minutes of the event. In some embodiments, S-2HG isadministered within 1 minute of the event. In some embodiments, S-2HG isadministered immediately after the occurance of the event. In someembodiments, the event is myocardial infarction, stroke, surgeries inwhich blood flow to a tissue is prevented such as in organtransplantations, and the like. In some embodiments, the event ismyocardial infarction.

In some embodiments, the therapeutically effective amount is about 5-50mg/kg, 5-40 mg/kg, 5-30 mg/kg, 5-20 mg/kg, 5-10 mg/kg, 10-40 mg/kg,10-30 mg/kg, 10-20 mg/kg, or about 10-15 mg/kg of the subject. In someembodiments, the therapeutically effective amount is about 5-20 mg/kg,or about 10-15 mg/kg of the subject. In some embodiments, thetherapeutically effective amount is about 5-50 mg/kg. In someembodiments, the therapeutically effective amount is about 5-40 mg/kg.In some embodiments, the therapeutically effective amount is about 5-30mg/kg. In some embodiments, the therapeutically effective amount isabout 5-20 mg/kg. In some embodiments, the therapeutically effectiveamount is about 5-10 mg/kg. In some embodiments, the therapeuticallyeffective amount is about 10-40 mg/kg. In some embodiments, thetherapeutically effective amount is about 10-30 mg/kg. In someembodiments, the therapeutically effective amount is about 10-20 mg/kg.In some embodiments, the therapeutically effective amount is about 10-15mg/kg.

In some embodiments, the compound of Formula I or Formula III isadministered as a single dose.

In some embodiments, the compound of Formula I or Formula III isadministered to the subject in combination with an additionaltherapeutic agent. In some embodiments, the additional therapeutic agentis oligomycin. In some embodiments, the additional therapeutic agent isaspirin.

In some embodiments, in the event that a method is for preventing orinhibiting an ischemia-reperfusion injury (IR injury) to a tissue in asubject, the compound of Formula I or Formula III is administered priorto an event that causes IR injury, reperfusion injury, or both. In someembodiments, the event is myocardial infarction, stroke, surgeries inwhich blood flow to a tissue is prevented such as in organtransplantations, and the like. In some embodiments, the event ismyocardial infarction. In some embodiments, the compound of Formula I orFormula III is α-KG. In some embodiments, the compound of Formula I orFormula III is a 2-HG compound. In some embodiments, the compound ofFormula I or Formula III is S-2HG.

In some embodiments, further disclosed herein is a method of increasingcyclophilin D (CypD)-Complex V formation comprising contacting a cellwith a compound of Formula III:

wherein: R¹ is hydrogen, halogen, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹,—SR¹⁰, substituted or unsubstituted alkyl, or substituted orunsubstituted heteroalkyl; R² and R³ are each independently hydrogen,halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl; or R² and R³, together with the atom to which they arebound, form an oxo; R⁴ and R⁵ are each independently hydrogen, halogen,—CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl; R⁶ ishalogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰,substituted or unsubstituted alkyl, or substituted or unsubstitutedheteroalkyl; and R⁷, R⁸, R⁹, and R¹⁰ are each independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; or a salt thereof. In someembodiments, R¹ is hydrogen, —CHO, or —OR⁷. In some embodiments, R¹ is—OR⁷, wherein R⁷ is hydrogen, substituted or unsubstituted alkyl, orsubstituted or unsubstituted aryl. In some embodiments, R¹ is —OR⁷,wherein R⁷ is C₁₋₂₀ substituted or unsubstituted alkyl. In someembodiments, R² is hydrogen, halogen, —CN, —CHO, or —NR⁸R⁹, wherein R⁸and R⁹ are each independently hydrogen or substituted or unsubstitutedalkyl. In some embodiments, R² and R³, together with the atom to whichthey are bound, form an oxo. In some embodiments, R⁴ and R⁵ are eachindependently hydrogen, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷, or —CONR⁸R⁹, whereinR⁷, R⁸, and R⁹ are each independently hydrogen or C₁₋₂₀ substituted orunsubstituted alkyl. In some embodiments, R⁶ is —CHO, —OR⁷, —NR⁸R⁹,—COOR⁷, or —CONR⁸R⁹, wherein R⁷, R⁸, and R⁹ are each independentlyhydrogen or C₁₋₂₀ substituted or unsubstituted alkyl. In someembodiments, R⁶ is —COOR⁷ or —CONR⁸R⁹, wherein R⁷, R⁸, and R⁹ are eachindependently hydrogen or C₁₋₂₀ substituted or unsubstituted alkyl. Insome embodiments, a compound of Formula III is alpha-ketoglutaric acid(α-KG). In some embodiments, a compound of Formula III is dimethyl2-oxoglutarate. In some embodiments, a compound of Formula III is a 2-HGcompound. In some embodiments, a compound of Formula III is2-hydroxyglutaric acid. In some embodiments, a compound of Formula IIIis S-2-hydroxyglutaric acid. In some embodiments, the increase incyclophilin D-Complex V formation is relative to the formation of CypDand Complex V of an equivalent cell in the absence of a compound ofFormula III. In some embodiments, the cell is from a cardiac tissue. Insome embodiments, the cell is from a mammalian cardiac tissue. In someembodiments, the cell is from a human cardiac tissue.

Additional Exemplary Pharmaceutical Compositions and Formulations

The one or more glutarate compounds to be administered to a subject maybe provided as a pharmaceutical composition or formulation.Pharmaceutical formulations may be prepared in a unit-dosage formappropriate for the desired mode of administration. The pharmaceuticalformulations of the present disclosure may be administered by anysuitable route including oral, rectal, nasal, topical (including buccaland sublingual), vaginal, and parenteral (including subcutaneous,intramuscular, intravenous, and intradermal). It will be appreciatedthat the route of administration may vary with the condition and age ofthe recipient, the nature of the condition to be treated, and the givencompound(s) of the present disclosure. In some embodiments, the route ofadministration is oral.

In some embodiments, pharmaceutical compositions disclosed hereincomprise a therapeutically effective amount of one or more compounds ofthe present disclosure, and a pharmaceutically acceptable carrier ordiluent. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial, and antifungal agents, isotonic and absorptiondelaying agents, stabilizers, diluents, suspending agents, thickeningagents, excipients, and the like, compatible with pharmaceuticaladministration. The pharmaceutical carrier employed may be either asolid or liquid.

Pharmaceutical compositions are optionally manufactured in aconventional manner, such as, by way of example only, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or compression processes.

In certain embodiments, compositions may also include one or more pHadjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In other embodiments, compositions may also include one or more salts inan amount required to bring osmolality of the composition into anacceptable range. Such salts include those having sodium, potassium orammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g., a compound described herein and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g., a compound described herein and a co-agent, areadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific intervening time limits,wherein such administration provides effective levels of the twocompounds in the body of the patient. The latter also applies tococktail therapy, e.g., the administration of three or more activeingredients.

The pharmaceutical compositions described herein are formulated into anysuitable dosage form, including but not limited to, aqueous oraldispersions, liquids, gels, syrups, elixirs, slurries, suspensions andthe like, for oral ingestion by an individual to be treated, solid oraldosage forms, aerosols, controlled release formulations, fast meltformulations, effervescent formulations, lyophilized formulations,tablets, powders, pills, dragees, capsules, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate releaseand controlled release formulations. In some embodiments, thecompositions are formulated into capsules. In some embodiments, thecompositions are formulated into solutions (for example, for IVadministration).

The pharmaceutical solid dosage forms described herein optionallyinclude a compound described herein and one or more pharmaceuticallyacceptable additives or excipients such as a compatible carrier, binder,filling agent, suspending agent, flavoring agent, sweetening agent,disintegrating agent, dispersing agent, surfactant, lubricant, colorant,diluent, solubilizer, moistening agent, plasticizer, stabilizer,penetration enhancer, wetting agent, anti-foaming agent, antioxidant,preservative, or one or more combination thereof.

In still other aspects, using standard coating procedures, such as thosedescribed in Remington's Pharmaceutical Sciences, 20th Edition (2000), afilm coating is provided around the compositions. In some embodiments,the compositions are formulated into particles (for example foradministration by capsule) and some or all of the particles are coated.In some embodiments, the compositions are formulated into particles (forexample for administration by capsule) and some or all of the particlesare microencapsulated. In some embodiments, the compositions areformulated into particles (for example for administration by capsule)and some or all of the particles are not microencapsulated and areuncoated.

In some embodiments, compositions provided herein may also include oneor more preservatives to inhibit microbial activity. Suitablepreservatives include mercury-containing substances such as merfen andthiomersal; stabilized chlorine dioxide; and quaternary ammoniumcompounds such as benzalkonium chloride, cetyltrimethylammonium bromideand cetylpyridinium chloride.

“Antifoaming agents” reduce foaming during processing which can resultin coagulation of aqueous dispersions, bubbles in the finished film, orgenerally impair processing. Exemplary anti-foaming agents includesilicon emulsions or sorbitan sesquoleate.

“Antioxidants” include, for example, butylated hydroxytoluene (BHT),sodium ascorbate, ascorbic acid, sodium metabisulfite and tocopherol. Incertain embodiments, antioxidants enhance chemical stability whererequired.

Formulations described herein may benefit from antioxidants, metalchelating agents, thiol containing compounds and other generalstabilizing agents. Examples of such stabilizing agents, include, butare not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/vmonothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% toabout 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate80, (g) 0.001% to about 0.05% w/v polysorbate 20, (h) arginine, (i)heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosanpolysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

“Binders” impart cohesive qualities and include, e.g., alginic acid andsalts thereof; cellulose derivatives such as carboxymethylcellulose,methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®),ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g.,Avicel®); microcrystalline dextrose; amylose; magnesium aluminumsilicate; polysaccharide acids; bentonites; gelatin;polyvinylpyrrolidone/vinyl acetate copolymer; crospovidone; povidone;starch; pregelatinized starch; tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), and lactose; a natural or synthetic gum suchas acacia, tragacanth, ghatti gum, mucilage of isapol husks,polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone®XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodiumalginate, and the like.

Examples of a “carrier” or “carrier materials” include excipients inpharmaceutics and should be selected on the basis of compatibility withcompounds disclosed herein, such as, compounds of Formula I or FormulaIII, and the release profile properties of the desired dosage form.Exemplary carrier materials include, e.g., binders, suspending agents,disintegration agents, filling agents, surfactants, solubilizers,stabilizers, lubricants, wetting agents, diluents, and the like.“Pharmaceutically compatible carrier materials” may include, but are notlimited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999).

“Dispersing agents,” and/or “viscosity modulating agents” includematerials that control the diffusion and homogeneity of a drug throughliquid media or a granulation method or blend method. In someembodiments, these agents also facilitate the effectiveness of a coatingor eroding matrix. Exemplary diffusion facilitators/dispersing agentsinclude, e.g., hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG,polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and thecarbohydrate-based dispersing agents such as, for example, hydroxypropylcelluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., PluronicsF68®, F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); and poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Corporation, Parsippany, N.J.)),polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetatecopolymer (S-630), polyethylene glycol, e.g., the polyethylene glycolcan have a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose can also be used as dispersing agents. Dispersingagents particularly useful in liposomal dispersions and self-emulsifyingdispersions are dimyristoyl phosphatidyl choline, natural phosphatidylcholine from eggs, natural phosphatidyl glycerol from eggs, cholesteroland isopropyl myristate.

Combinations of one or more erosion facilitator with one or morediffusion facilitator can also be used in the present compositions.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution. In certain embodiments,diluents increase bulk of the composition to facilitate compression orcreate sufficient bulk for homogenous blend for capsule filling. Suchcompounds include e.g., lactose, starch, mannitol, sorbitol, dextrose,microcrystalline cellulose such as Avicel®; dibasic calcium phosphate,dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate;anhydrous lactose, spray-dried lactose; pregelatinized starch,compressible sugar, such as Di-Pac® (Amstar); mannitol,hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetatestearate, sucrose-based diluents, confectioner's sugar; monobasiccalcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactatetrihydrate, dextrates; hydrolyzed cereal solids, amylose; powderedcellulose, calcium carbonate; glycine, kaolin; mannitol, sodiumchloride; inositol, bentonite, and the like.

The term “disintegrate” includes both the dissolution and dispersion ofthe dosage form when contacted with gastrointestinal fluid.“Disintegration agents or disintegrants” facilitate the breakup ordisintegration of a substance. Examples of disintegration agents includea starch, e.g., a natural starch such as corn starch or potato starch, apregelatinized starch such as National 1551 or Amijel®, or sodium starchglycolate such as Promogel® or Explotab®, a cellulose such as a woodproduct, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101,Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, MingTia®, and Solka-Floc®, methylcellulose, croscarmellose, or across-linked cellulose, such as cross-linked sodiumcarboxymethylcellulose (Ac-Di-Sol®), cross-linkedcarboxymethylcellulose, or cross-linked croscarmellose, a cross-linkedstarch such as sodium starch glycolate, a cross-linked polymer such ascrospovidone, a cross-linked polyvinylpyrrolidone, alginate such asalginic acid or a salt of alginic acid such as sodium alginate, a claysuch as Veegum® HV (magnesium aluminum silicate), a gum such as agar,guar, locust bean, Karaya, pectin, or tragacanth, sodium starchglycolate, bentonite, a natural sponge, a surfactant, a resin such as acation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium laurylsulfate in combination starch, and the like.

“Drug absorption” or “absorption” typically refers to the process ofmovement of drug from site of administration of a drug across a barrierinto a blood vessel or the site of action, e.g., a drug moving from thegastrointestinal tract into the portal vein or lymphatic system.

An “enteric coating” is a substance that remains substantially intact inthe stomach but dissolves and releases the drug in the small intestineor colon. Generally, the enteric coating comprises a polymeric materialthat prevents release in the low pH environment of the stomach but thationizes at a higher pH, typically a pH of 6 to 7, and thus dissolvessufficiently in the small intestine or colon to release the active agenttherein.

“Erosion facilitators” include materials that control the erosion of aparticular material in gastrointestinal fluid. Erosion facilitators aregenerally known to those of ordinary skill in the art. Exemplary erosionfacilitators include, e.g., hydrophilic polymers, electrolytes,proteins, peptides, and amino acids.

“Filling agents” include compounds such as lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol,mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

“Flavoring agents” and/or “sweeteners” useful in the formulationsdescribed herein, include, e.g., acacia syrup, acesulfame K, alitame,anise, apple, aspartame, banana, Bavarian cream, berry, black currant,butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream,chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream,cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate,cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger,glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey,isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate(MagnaSweet®), maltol, mannitol, maple, marshmallow, menthol, mintcream, mixed berry, neohesperidine DC, neotame, orange, pear, peach,peppermint, peppermint cream, Prosweet® Powder, raspberry, root beer,rum, saccharin, safrole, sorbitol, spearmint, spearmint cream,strawberry, strawberry cream, stevia, sucralose, sucrose, sodiumsaccharin, saccharin, aspartame, acesulfame potassium, mannitol, talin,sylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine,thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry,wintergreen, xylitol, or any combination of these flavoring ingredients,e.g., anise-menthol, cherry-anise, cinnamon-orange, cherry-cinnamon,chocolate-mint, honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus,orange-cream, vanilla-mint, and mixtures thereof.

“Lubricants” and “glidants” are compounds that prevent, reduce orinhibit adhesion or friction of materials. Exemplary lubricants include,e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, ahydrocarbon such as mineral oil, or hydrogenated vegetable oil such ashydrogenated soybean oil (Sterotex®), higher fatty acids and theiralkali-metal and alkaline earth metal salts, such as aluminum, calcium,magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes,Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

A “measurable serum concentration” or “measurable plasma concentration”describes the blood serum or blood plasma concentration, typicallymeasured in mg, μg, or ng of therapeutic agent per mL, dL, or L of bloodserum, absorbed into the bloodstream after administration. As usedherein, measurable plasma concentrations are typically measured in ng/mlor μg/ml.

“Pharmacodynamics” refers to the factors which determine the biologicresponse observed relative to the concentration of drug at a site ofaction.

“Pharmacokinetics” refers to the factors which determine the attainmentand maintenance of the appropriate concentration of drug at a site ofaction.

“Plasticizers” are compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. In someembodiments, plasticizers can also function as dispersing agents orwetting agents.

“Solubilizers” include compounds such as triacetin, triethylcitrate,ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

“Stabilizers” include compounds such as any antioxidation agents,buffers, acids, preservatives and the like.

“Steady state,” as used herein, is when the amount of drug administeredis equal to the amount of drug eliminated within one dosing intervalresulting in a plateau or constant plasma drug exposure.

“Suspending agents” include compounds such as polyvinylpyrrolidone,e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g.,the polyethylene glycol can have a molecular weight of about 300 toabout 6000, or about 3350 to about 4000, or about 7000 to about 5400,sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

“Surfactants” include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Someother surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. In some embodiments, surfactants may be included toenhance physical stability or for other purposes.

“Viscosity enhancing agents” include, e.g., methyl cellulose, xanthangum, carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetatestearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinylalcohol, alginates, acacia, chitosans and combinations thereof.

“Wetting agents” include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

EXAMPLES

The following examples are intended to illustrate but not to limit theinvention.

Example 1 Post-Ischemic Application of α-KG Rapidly Restores HeartHemodynamic Parameters Ex Vivo

The protective effect of α-KG on heart hemodynamic parameters in ex vivomodel of ischemia-reperfusion injury (IR injury) was examined. IsolatedLangendorff perfused hearts were subjected to 30 minutes ischemiafollowed by 60 minutes reperfusion with Krebs Henseleit (KH) buffercontaining 800 μM α-KG or with KH buffer as control (FIG. 1).Administration of α-KG during reperfusion rapidly restored the heartfunction within the first few minutes of reperfusion as the leftventricle developed pressure (LVDP) was much higher compared to controltreatment (FIG. 2, FIG. 3, and FIG. 4). Rate pressure product (RPP),which is calculated as LVDP×heart rate, was also significantly higher inα-KG with about 70% recovery observed after 5 minutes of reperfusion vs.5% in the control group, and about 80% after 30 minutes of reperfusionin α-KG compared to only 20% in control group (FIG. 5). The α-KG groupalso showed a much better LV dP/dt_(max) compared to CTRL hearts (FIG.6).

Because the first few minutes of reperfusion are critical in myocardialprotection. These results suggest that α-KG could serve as a noveltherapeutic agent for targeting the critical first few minutes ofreperfusion to rapidly restore the heart function and reduce infarctsize. Indeed, this was confirmed by the following in vivo experiments.

Example 2 Administration of α-KG at the Onset of Reperfusion ReducesMyocardial Infarct Size In Vivo

The protective role of α-KG in in vivo mouse model of IR injury, whichis a close representation of the intact heart, was examined. The leftcoronary artery was occluded for 30 minutes followed by 24 hours ofreperfusion (FIG. 7). One single bolus of α-KG (800 μM, unoptimized)given through the tail vein at the onset of reperfusion reduced theinfarct size in mice by about 70% (FIG. 8 and FIG. 9). The smallerinfarct size in α-KG group was not due to smaller AAR (area at risk)since both groups were subjected to a comparable degree of ischemicrisk. Further experiments show that 200 μM of α-KG confers similarlevels of protection as 800 μM of α-KG. These data demonstrate a robustprotective role of α-KG against IR injury in vivo.

Example 3 Role of Complex V Inhibition in Protection

As provided in FIG. 10, α-KG inhibits complex V activity in mitochondriaisolated from mouse heart at the baseline. The mitochondrial F₁F_(o) ATPsynthase is the main source of ATP in mammals. The ATP synthase canswitch to an ATP hydrolase under certain conditions, includingmyocardial ischemia, resulting in the loss of as much as 50-90% of thetotal ATP. Experiments were conducted to determine whether oligomycincan alleviate IR injury when administered at the time of reperfusion.Oligomycin was found to be toxic at 5 μM, however, lower doses (e.g., 10nM) of oligomycin given at reperfusion were found to significantlyreduce myocardial infarct size in mice (FIG. 8 and FIG. 9). Theseresults are unexpected and suggest that inhibition of ATP synthase is atleast partially responsible for the protective effect of α-KG. Sinceoligomycin is considered unsafe for treating IR injury due to concernsof poisoning ATP production in healthy tissue and α-KG is a naturallyoccurring metabolite having proven safety profiles and is more effectivein reducing IR injury, α-KG alone or in combination with low doses ofoligomycin can be used to treat or reduce IR injury.

Example 4 Protection by α-KG is Independent of HIF, PHD, and Epigenetics

Since HIF-1 protein stability is regulated by prolyl hydroxylase domaincontaining proteins (PHD; encoded by Egl nine homolog or EGLN genes) forwhich α-KG is a cofactor (as is oxygen), it was surprising to find thatneither HIF-1 nor PHD is involved in protection by α-KG. Not only isα-KG fully protective in the presence of the HIF-1 inhibitorechinomycin, but the PHD inhibitor (S)-2-hydroxyglutarate (S-2HG)confers similar extents of protection as α-KG (FIG. 12). Therefore, α-KGand/or S-2HG can be used to treat or reduce IR injury.

In addition to PHD in hypoxic response, many other dioxygenases also useα-KG as a co-substrate in a variety of cellular processes includingepigenetic regulation. Because the protective effect of α-KG manifestswithin minutes of injection as shown by ex vivo experiments herein, andan inhibitor of α-KG-dependent epigenetic enzymes (S-2HG) also conferssimilar extents of protection against IR injury in vivo, epigeneticmechanisms can largely be ruled out.

Example 5 Protection by an α-KG Metabolite, Succinyl-CoA, Against IRInjury

In the cell, α-KG is decarboxylated to succinyl-CoA and CO₂ by α-KGdehydrogenase (α-KGDH). Interestingly, it was found that succinyl-CoAexhibits significant protection against IR injury, whereas succinate,which is the next intermediate in the TCA cycle, does not (FIG. 13).These results indicate that succinyl-CoA itself plays an active role inprotection and open a new area of investigation into additionaltherapeutic strategies against IR injury.

Example 6 α-KG Increases Cyclophilin D Binding to Complex V

Mitochondrial membrane permeability transition pore (mPTP) is astructure that forms in the inner membrane of the mitochondria undercertain pathological conditions including stroke and myocardialischemia-reperfusion. Induction of mPTP leads to mitochondrial swelling,loss of membrane potential, and activation of cell death signaling. mPTPconsists of Complex V and cyclophilin D (CypD). The opening of mPTP isaffected by the binding affinity of CypD to Complex V; increased CypDbinding inhibits the induction of mPTP. In some embodiments, studieshave shown that mPTP opening can be induced or inhibited by Complex Vinhibitors. See e.g., Giorgio, et al. (2013) PNAS USA 110:5887-5892.

As illustrated in Example 3, α-KG inhibits the F₁ subunit of Complex V.In this example, it was found that α-KG increased CypD binding toComplex V (FIG. 14). This suggests that α-KG may inhibit the inductionof mPTP opening during reperfusion to confer cardioprotection. The knownComplex V inhibitor oligomycin (which inhibits the Fo subunit ofComplexV) also increases CypD-Complex V affinity and conferscardioprotection. In contrast, Bz-423, another Complex V inhibitor,reduces CypD affinity to Complex V and induces apoptosis.

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified.

The section headings used herein are for organizational purposes and arenot to be construed as limiting the subject matter described.

As used herein, the terms “subject”, “patient”, and “individual” areused interchangeably to refer to humans and non-human animals. The term“non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, horses, sheep, dogs, cows,pigs, chickens, and other veterinary subjects and test animals. In someembodiments, the subject is a mammal. In some embodiments, the subjectis a human.

The use of the singular can include the plural unless specificallystated otherwise. As used in the specification and the appended claims,the singular forms “a”, “an”, and “the” can include plural referentsunless the context clearly dictates otherwise. The use of “or” can mean“and/or” unless stated otherwise. As used herein, “and/or” means “and”or “or”. For example, “A and/or B” means “A, B, or both A and B” and “A,B, C, and/or D” means “A, B, C, D, or a combination thereof” and said“combination thereof” means any subset of A, B, C, and D, for example, asingle member subset (e.g., A or B or C or D), a two-member subset(e.g., A and B; A and C; etc.), or a three-member subset (e.g., A, B,and C; or A, B, and D; etc.), or all four members (e.g., A, B, C, andD).

To the extent necessary to understand or complete the disclosure of thepresent invention, all publications, patents, and patent applicationsmentioned herein are expressly incorporated by reference therein to thesame extent as though each were individually so incorporated.

Having thus described exemplary embodiments of the present invention, itshould be noted by those skilled in the art that the within disclosuresare exemplary only and that various other alternatives, adaptations, andmodifications may be made within the scope of the present invention.Accordingly, the present invention is not limited to the specificembodiments as illustrated herein, but is only limited by the followingclaims.

1-20. (canceled)
 21. A method of treating or reducing anischemia-reperfusion (IR) injury to a tissue in a subject, comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising: a therapeutically effectiveamount of a compound of Formula II:

or a salt thereof, wherein: R¹ is hydrogen, halogen, —CHO, —OR⁷, —NR⁸R⁹,—COOR⁷, —CONR⁸R⁹, —SR¹⁰, substituted or unsubstituted alkyl, orsubstituted or unsubstituted heteroalkyl; R² and R³ are eachindependently hydrogen, halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷,—CONR⁸R⁹, —NO₂, —SR¹⁰, substituted or unsubstituted alkyl, orsubstituted or unsubstituted heteroalkyl; or R² and R³, together withthe atom to which they are bound, form an oxo; R⁴ and R⁵ are eachindependently hydrogen, halogen, —CN, —CHO, —OR⁷, —NR⁸R⁹, —COOR⁷,—CONR⁸R⁹, —NO₂, —SR¹⁰, substituted or unsubstituted alkyl, orsubstituted or unsubstituted heteroalkyl; R⁶ is halogen, —CN, —CHO,—OR⁷, —NR⁸R⁹, —COOR⁷, —CONR⁸R⁹, —NO₂, —SR¹⁰, substituted orunsubstituted alkyl, or substituted or unsubstituted heteroalkyl; andeach R⁷, R⁸, R⁹, and R¹⁰ is independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; wherein each R⁷ substituent is independentlyselected when two or more R⁷ substituents are present, each R⁸substituent is independently selected when two or more R⁸ substituentsare present, each R⁹ substituent is independently selected when two ormore R⁹ substituents are present, and each R¹⁰ substituent isindependently selected when two or more R¹⁰ substituents are present;wherein the pharmaceutical composition is administered to the subject totreat or reduce IR injury to a tissue in the subject; and wherein thetherapeutically effective amount is about 5-20 mg of the compound ofFormula II, or the salt thereof, per kilogram weight of the subject. 22.The method of claim 21, wherein R¹ is —OH, —O-(C₁₋₂₀ substituted alkyl)or —O-(C₁₋₂₀ unsubstituted alkyl).
 23. The method of claim 22, whereinR¹ is —O-(n—octyl).
 24. The method of claim 22, wherein R¹ is —OH. 25.The method of claim 21, wherein R² is —OR⁷ or —NR⁸R⁹.
 26. The method ofclaim 25, wherein R² is —OH or —NH₂.
 27. The method of claim 21, whereinR³ is hydrogen.
 28. The method of claim 21, wherein R² and R³, togetherwith the atom to which they are bound, form an oxo.
 29. The method ofclaim 21, wherein R⁴ and R⁵ are each independently hydrogen or —CH₃. 30.The method of claim 31, wherein R⁶ is —COOH, —COO-(C₁₋₂₀ unsubstitutedalkyl), —CONH₂, or —CONH-(C₁₋₂₀ unsubstituted alkyl).
 31. The method ofclaim 30, wherein R⁶ is —COOH or —COO-(n-octyl).
 32. The method of claim21, wherein: R¹ is hydrogen, —OH, —O-(C₁₋₂₀ substituted alkyl) or—O-(C₁₋₂₀ unsubstituted alkyl); R² and R³ are each independentlyhydrogen, —OR⁷, or —NR⁸R⁹; or R² and R³ form an oxo together with theatom to which they are bound; R⁴ and R⁵ are each independently hydrogenor —CH₃; and R⁶ is —COOH, —COO-(C₁₋₂₀ unsubstituted alkyl), —COO-(C₁₋₂₀substituted alkyl), CONH₂, —CONH-(C₁₋₂₀ unsubstituted alkyl), or—CONH-(C₁₋₂₀ substituted alkyl).
 33. The method of claim 32, wherein R¹is —OH and R⁶ is —COO-(C₁₋₂₀ unsubstituted alkyl); or R¹ is —O-(C₁₋₂₀unsubstituted alkyl) and R⁶ is —COOH.
 34. The method of claim 21,wherein the compound of Formula II is administered during or after anevent that causes IR injury or reperfusion injury.
 35. The method ofclaim 34, wherein the compound of Formula II is administered within 1 to2 hours of the event.
 36. The method of claim 34, wherein the event is amyocardial infarction.
 37. The method of claim 21, wherein the compoundof Formula II is administered as a single dose.
 38. The method of claim21, wherein the compound of Formula II is administered to the subject incombination with an additional therapeutic agent.
 39. The method ofclaim 38, wherein the additional therapeutic agent is oligomycin oraspirin.
 40. The method of claim 21, wherein the tissue is cardiactissue.